Effect of pooled tracheal sample testing on the probability of Mycoplasma hyopneumoniae detection.

Tracheal pooling for Mycoplasma hyopneumoniae (M. hyopneumoniae) DNA detection allows for decreased diagnostic cost, one of the main constraints in surveillance programs. The objectives of this study were to estimate the sensitivity of pooled-sample testing for the detection of M. hyopneumoniae in tracheal samples and to develop probability of M. hyopneumoniae detection estimates for tracheal samples pooled by 3, 5, and 10. A total of 48 M. hyopneumoniae PCR-positive field samples were pooled 3-, 5-, and 10-times using field M. hyopneumoniae DNA-negative samples and tested in triplicate. The sensitivity was estimated at 0.96 (95% credible interval [Cred. Int.]: 0.93, 0.98) for pools of 3, 0.95 (95% Cred. Int: 0.92, 0.98) for pools of 5, and 0.93 (95% Cred. Int.: 0.89, 0.96) for pools of 10. All pool sizes resulted in PCR-positive if the individual tracheal sample Ct value was < 33. Additionally, there was no significant decrease in the probability of detecting at least one M. hyopneumoniae-infected pig given any pool size (3, 5, or 10) of tracheal swabs. Furthermore, this manuscript applies the probability of detection estimates to various real-life diagnostic testing scenarios. Combining increased total animals sampled with pooling can be a cost-effective tool to maximize the performance of M. hyopneumoniae surveillance programs.

Feed or feed transport as a potential route for a porcine epidemic diarrhoea outbreak in a 10,000-sow breeding herd in Mexico.

Porcine epidemic diarrhoea virus (PEDV) infects pigs of all ages causing vomiting and diarrhoea. PEDV is transmitted via the oral-faecal route, and a very low dose is enough to infect susceptible pigs, resulting in significant production losses. This short communication aims to describe the introduction of PEDV into a 10,000-sow farrow-to-wean farm located in northwest Mexico. Following the onset of clinical signs, an outbreak investigation was conducted to determine the most probable route of introduction. Based on data collected from interviews, construction of a timeline of events, and the detection of PEDV RNA in feed samples and samples collected from various surfaces of feed transport vehicles, it was concluded that the most probable route for PEDV incursion into this breeding herd was contaminated feed or a contaminated feed transport vehicle. This paper describes how feed or feed transport could serve as potential routes of PEDV infection to a farm and highlights the importance of establishing biosecurity programs to mitigate these risks.

Probability of PRRS virus detection in pooled processing fluid samples.

There has been a tremendous increase in recent years of population-based diagnostic monitoring and surveillance strategies in swine populations. One example is the use of processing fluids (PF) to screen breeding herds for porcine reproductive and respiratory syndrome virus (PRRSV) activity. An important question from practitioners using such methods is on how intensively can the sample be pooled. More specifically, processing fluids of how many litters can be pooled into a single sample for diagnostic testing to preserve a high probability of PRRSV RNA detection at low prevalence situations? The objective of this study was to model the effect of pooling PF samples on the probability of PRRSV RNA detection. For this study, a PRRSV-positive PF field sample with a RT-rtPCR quantification cycle (Cq) value of 28 was selected to represent a litter of 11 pigs with a single viremic piglet. PF samples from a PRRSV-naïve herd were used to perform 6 replications of 8 two-fold serial dilutions of the PRRSV-positive sample, thus modeling the pooling effect (dilution). Each two-fold dilution represented an increase in the number of PRRS-negative pigs in the sample by a factor of 2. Samples were tested for PRRSV RNA by RT-rtPCR and the data was analyzed using linear and probit regression models. There was an average increment of 1.37 points in Ct for each two-fold dilution. The estimated probability of testing positive on RT-rtPCR was 43 %, 80 %, and 95 % when there was a single PRRSv-positive piglet among 784, 492, and 323 PRRSv-negative piglets contributing to the sample respectively. Results from this study support the practice of collecting and aggregating PF samples from multiple litters for PRRSV RNA testing.

Mycoplasma hyopneumoniae Surveillance in Pig Populations: Establishing Sampling Guidelines for Detection in Growing Pigs.

Antemortem detection of Mycoplasma hyopneumoniae infection in swine production systems has relied on antibody testing, but the availability of tests based on DNA detection and novel diagnostic specimens, e.g., tracheal swabs and oral fluids, has the potential to improve M. hyopneumoniae surveillance. A field study was performed over a 14-week period during which 10 pigs in one pen at the center of a room with 1,250 6-week-old pigs housed in 46 pens were intratracheally inoculated with M. hyopneumoniae Thereafter, one tracheal sample, four serum samples, and one oral fluid sample were collected from every pen at 2-week intervals. Tracheal and oral fluid samples were tested for M. hyopneumoniae DNA and serum samples for M. hyopneumoniae antibody. Test results were modeled using a hierarchical Bayesian model, based on a latent spatial piecewise exponential survival model, to estimate the probability of detection by within-pen prevalence, number of positive pens in the barn, sample allocation, sample size, and sample type over time. Analysis showed that tracheal samples provided the earliest detection, especially at large sample sizes. While serum samples are more commonly collected and are less expensive to test, high probability of detection estimates were only obtained 30 days postexposure at large sample sizes. In all scenarios, probability of detection estimates for oral fluids within 30 days were significantly lower than those for tracheal and serum samples. Ultimately, the choice of specimen type, sample number, and assay will depend on testing objectives and economics, but the estimates provided here will assist in the design of M. hyopneumoniae surveillance and monitoring programs for different situations.

Practical aspects of PRRSV RNA detection in processing fluids collected in commercial swine farms.

Processing fluid samples are easily collected under field conditions and provide the means to test more piglets more frequently in a practical way, thereby improving PRRSV surveillance. However, a deeper understanding of the diagnostic characteristics of this newly described sample type is still required. Therefore, the objective of this field-based study was to determine the relationship between viremic piglets and the detection of PRRSV RNA in processing fluid samples. In two PRRSV-positive breeding herds, processing fluids (n = 77) and individual piglet serum samples (n = 834) were collected from 77 litters in three sampling events and tested for PRRSV RNA. Among the 77 litters in the study, 55 litters (71.4%) contained no viremic piglets and processing fluids tested negative for PRRSV RNA. Among the 22 (28.6%) litters with ≥1 viremic piglets, 10 litters contained a single viremic piglet and 5 of the 10 processing fluids from this group tested positive for PRRSV RNA. Based on a fitted mixed effects logistic regression model, the probability of detecting PRRSV RNA in processing fluids was highly dependent on the number of viremic piglets contributing to the sample. When the within-litter prevalence was ≥39%, the probability of detecting PRRSV RNA in processing fluids was ≥95%. By extension, the results suggest that pooling processing fluids from several litters increases the probability of PRRSV RNA detection because of the greater likelihood of including multiple litters each with ≥1 viremic piglets. In contemporary breeding herds that use processing fluid samples for PRRSV surveillance, the diagnostic costs associated with testing 100% of the processing-age piglet population can be estimated at €0.077 ($0.086 USD) per pig weaned. In contrast, to achieve an equivalent testing coverage with the use of individual piglet serum samples, the diagnostic costs associated would be €4.48 ($5.00 USD) per pig weaned. Processing fluid represents a practical, reliable and efficient method to surveil breeding herds for PRRSV because it allows for continuous surveillance at a low cost.

Comparison of individual, group and environmental sampling strategies to conduct influenza surveillance in pigs.

BACKGROUND: Influenza A virus (IAV) is an important pathogen in pigs that affects productivity and has important public health implications because of its zoonotic nature. Surveillance is central to the control of influenza, however, detection of IAV infections can be challenging in endemically infected herds with low prevalence of infection. METHODS: In groups of suckling (18-21 days of age) and growing (35-45 days of age) pigs, we compared various sampling approaches to detect, isolate and sequence IAV using individual (nasal swabs, nasal wipes and oropharyngeal swabs), group (oral fluids, surface wipes and sow udder skin wipes) and environmental (airborne particles deposited on surfaces and air samples) sampling approaches. All samples were tested by IAV rRT-PCR and a subset was used for virus isolation and direct sequencing. RESULTS: In general, environmental and group samples resulted in higher odd ratios (range = 3.87-16.5, p-value < 0.05) of detecting a positive sample by rRT-PCR compared to individual pooled samples, except for oropharyngeal swabs (OR = 8.07, p-value < 0.05). In contrast, individual samples were most likely to yield a viral isolate by cell culture. Oropharyngeal swabs in suckling pigs (78.4%), and nasal swabs (47.6%) or nasal wipes (45%) in growing pigs, and udder wipes in lactating sows (75%) were the preferred samples to obtain an isolate. CONCLUSIONS: Our findings indicate that group and environmental sampling strategies should be considered in influenza surveillance programs in particular if the goal is just to detect infection. This study provides new information on sampling approaches to conduct effective influenza surveillance in pigs and identifies udder wipes from lactating sows as a novel sample type that offers a convenient, cheap and sensitive manner to monitor IAV in litters prior to weaning.

Stochastic model of porcine reproductive and respiratory syndrome virus control strategies on a swine farm in the United States.

OBJECTIVE: To use mathematical modeling to assess the effectiveness of control strategies for porcine reproductive and respiratory syndrome (PRRS) virus on a swine farm. SAMPLE: A hypothetical small, medium, or large farrow-to-weaning swine farm in the Midwestern United States. PROCEDURES: Stochastic models were formulated to simulate an outbreak of PRRS on a farm. Control strategies assessed in those models included none (baseline) and various combinations of mass immunization, herd closure, and gilt acclimatization. Nine different models resulting from the combination of low, moderate, or high PRRS virus virulence and small, medium, or large herd size were simulated. A stabilized status, the outcome of interest, was defined as the absence of positive PCR assay results for PRRS virus in 3-week-old piglets. For each scenario, the percentage of simulations with a stabilized status was used as a proxy for the probability of disease control. RESULTS: Increasing PRRS virus virulence and herd size were negatively associated with the probability of achieving a stabilized status. Repeated mass immunization with herd closure or gilt acclimitization was a better alternative than was single mass immunization for disease control within a farm. CONCLUSIONS AND CLINICAL RELEVANCE: Repeated mass immunization with a PRRS modified-live virus vaccine with herd closure or gilt acclimitization was the scenario most likely to achieve a stabilized status. Estimation of the cost of various PRRS control strategies is necessary.

Evaluation of the long-term effect of air filtration on the occurrence of new PRRSV infections in large breeding herds in swine-dense regions.

Airborne transmission of porcine reproductive and respiratory syndrome virus (PRRSV) is a risk factor for the infection of susceptible populations. Therefore, a long­term sustainability study of air filtration as a means to reduce this risk was conducted. Participating herds (n = 38) were organized into 4 independent cohorts and the effect of air filtration on the occurrence of new PRRSV infections was analyzed at 3 different levels from September 2008 to January 2012 including the likelihood of infection in contemporary filtered and non-filtered herds, the likelihood of infection before and after implementation of filtration and the time to failure in filtered and non-filtered herds. Results indicated that new PRRSV infections in filtered breeding herds were significantly lower than in contemporary non-filtered control herds (P < 0.01), the odds for a new PRRSV infection in breeding herds before filtration was 7.97 times higher than the odds after filtration was initiated (P < 0.01) and the median time to new PRRSV infections in filtered breeding herds of 30 months was significantly longer than the 11 months observed in non-filtered herds (P < 0.01). In conclusion, across all 3 levels of analysis, the long-term effect of air filtration on reducing the occurrence of new PRRSV infections in the study population was demonstrated.

Effect of modified-live porcine reproductive and respiratory syndrome virus (PRRSv) vaccine on the shedding of wild-type virus from an infected population of growing pigs.

There are ongoing efforts to eliminate porcine reproductive and respiratory syndrome virus (PRRSv) from regions in the United States swine industry. However, an important challenge for the accomplishment of those efforts is the re-infection of pig units due to the area spread of PRRSv. The objective of this study was to evaluate the effect of PRRS modified-live virus vaccine (MLV) on viral shedding and on dynamics of PRRSv infection in pig populations raised under commercial conditions. The study composed of two rooms of 1000 pigs each. Ten percent of pigs of each room were inoculated with a field isolate of PRRSv. Rooms had separate air spaces and strict scientifically validated biosecurity protocols were adopted to avoid movement of pathogens between rooms. At 8 and 36 dpi (days post inoculation), all pigs of the challenge-vaccine group were inoculated with a MLV vaccine. Pigs of the challenge-control group were placebo-inoculated. Blood and oral fluid samples were collected from each room at 0, 8, 36, 70, 96 and 118 dpi for PRRSv RNA detection using PCR. PRRSv-antibodies were also screened from blood serum samples with a commercially available ELISA test. Additionally, tonsil scraping samples were collected from both groups at 70, 96 and 118 dpi. Moreover, air samples were collected 6 times per week from 0 to 118 dpi and were tested for PRRSv RNA using qPCR assay. There was no difference in the PRRSv infection dynamics measured as duration and magnitude of viremia and seroconversion. Also, there was no difference in the frequency of tonsil scraping samples PRRSv-positive by PCR. However, the challenge-vaccine group had significantly less PRRSv shed compared to the challenge-control group. The challenge-vaccine group had significant less PRRSv-positive oral fluids at 36 dpi. Moreover, the challenge-vaccine group had significant reduction in the cumulative PRRSv shed in the air.

Reducing the diffraction artifacts while implementing a phase function on a spatial light modulator.

Spatial light modulators are often used to implement phase modulation. Since they are pixelated, the phase function is usually approximated by a regularly sampled piecewise constant function, and the periodicity of the pixel sampling generates annoying diffraction peaks. We theoretically investigate two pixelation techniques: the isophase method and a new nonperiodic method derived from the Voronoi tessellation technique. We show that, for a suitable choice of parameters, the diffraction peaks disappear and are replaced by a smoothly varying halo. We illustrate the potential of these two techniques for implementing a lens function and wavefront correction.

Minimization of diffraction peaks of spatial light modulators using Voronoi diagrams.

It is possible to reduce the diffraction peaks of a Spatial Light Modulator (SLM) by breaking the periodicity of the pixels shape. We propose a theoretical investigation of a SLM that would be based on a Voronoi diagram, obtained by deforming a regular grid, and show that for a specific deformation parameter the diffraction peaks disappear and are replaced with a speckle-like diffraction halo. We also develop a simple model to determine the shape and the level of this halo.

Infection dynamics and clinical manifestations following experimental inoculation of gilts at 90 days of gestation with a low dose of porcine reproductive and respiratory syndrome virus.

Understanding the dynamics of porcine reproductive and respiratory syndrome virus (PRRSV) vertical transmission is important to enhance the accuracy of monitoring protocols for endemically infected breeding herds. The objectives of this study were to determine the prevalence of PRRSV within infected litters, to quantify viremia, and to identify specific attributes of infected individuals. Eight gilts were intramuscularly inoculated with 10(1) TCID(50) of a mildly virulent PRRSV strain (MN-30100) at 90 d of gestation. All inoculated gilts transmitted the virus in utero. The proportion of PRRSV PCR-positive piglets and the level of viremia in the piglets were higher at 4 d of age than at birth or at weaning. No specific attributes were associated with PRRSV infection in the piglets. This is the first report, that we are aware of, documenting the efficient in utero transmission of an extremely low dose of a mildly virulent strain of PRRSV. The results support the sampling of piglets late during lactation as a tool to monitor PRRSV shedding from sow-herds.

Feasibility of pooled-sample testing for the detection of porcine reproductive and respiratory syndrome virus antibodies on serum samples by ELISA.

Surveillance of porcine reproductive and respiratory syndrome (PRRS) in negative sow farms is usually performed by testing for the presence of antibodies against PRRS virus in serum with a commercial ELISA test. The objective of this study was to evaluate the feasibility of pooling serum samples for detection of PRRS virus antibodies by ELISA. The effect of pool size on the sensitivity and specificity of the ELISA test was evaluated by testing true positive samples and false positive samples, respectively, diluted in negative sera. The effect of three different cut-off values for the interpretation of the diagnostic test (0.4, 0.3 and 0.2) was evaluated as well. Furthermore, the obtained sensitivity and specificity estimates were used to calculate the herd sensitivity and herd specificity of surveillance protocols in different scenarios. The results showed that pooling serum samples to detect PRRSV antibodies resulted in a decrease in sensitivity and an increase in specificity, compared to testing individual samples, while the reduction of the s/p cut-off value recommended by the manufacturer (0.4) had the opposite effect. We describe an approach that can increase the herd sensitivity of a surveillance protocol for breeding herds, while maintaining high herd specificity and low testing costs. This can be achieved by sampling a larger number of animals and running the samples in pools. Therefore, the conventional monitoring protocols based on ELISA on individual samples can be improved by using pooled-sample testing.

Antigen-specific B-cell responses to porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an acute, viremic infection of 4 to 6 weeks, followed by a persistent infection lasting for several months. We characterized antibody and B-cell responses to viral proteins in acute and persistent infection to better understand the immunological basis of the prolonged infection. The humoral immune response to PRRSV was robust overall and varied among individual viral proteins, with the important exception of a delayed and relatively weak response to envelope glycoprotein 5 (GP5). Memory B cells were in secondary lymphoid organs, not in bone marrow or Peyer's patches, in contrast to the case for many mammalian species. Potent anti-PRRSV memory responses were elicited to recall antigen in vitro, even though a second infection did not increase the B-cell response in vivo, suggesting that productive reinfection does not occur in vivo. Antibody titers to several viral proteins decline over time, even though abundant antigen is known to be present in lymphoid tissues, possibly indicating ineffective antigen presentation. The appearance of antibodies to GP5 is delayed relative to the resolution of viremia, suggesting that anti-GP5 antibodies are not crucial for resolving viremia. Lastly, viral infection had no immunosuppressive effect on the humoral response to a second, unrelated antigen. Taking these data together, the active effector and memory B-cell responses to PRRSV are robust, and over time the humoral immune response to PRRSV is effective. However, the delayed response against GP5 early in infection may contribute to the prolonged acute infection and the establishment of persistence.

Effect of vaccination with a modified-live porcine reproductive and respiratory syndrome virus vaccine on dynamics of homologous viral infection in pigs.

OBJECTIVE: To determine effects of vaccination protocols with modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on persistence and transmission of virus in pigs infected with a homologous isolate and determine clinical and virologic responses following heterologous viral challenge. ANIMALS: Four hundred forty 6- to 8-week-old PRRSV-naïve pigs. PROCEDURES: Pigs were allocated into 5 groups. Groups A to D were inoculated with wild-type PRRSV VR2332. Group A (positive control pigs) received PRRSV only. Groups B, C, and D received modified-live PRRSV vaccine (1, 2, or 3 doses). Group E served as a negative control group. To evaluate viral transmission, sentinel pigs were introduced into each group at intervals from 37 to 67, 67 to 97, and 97 to 127 days postinoculation (DPI). To evaluate persistence, pigs were euthanized at 37, 67, 97, or 127 DPI. To assess clinical and virologic response after challenge, selected pigs from each group were inoculated at 98 DPI with a heterologous isolate (PRRSV MN-184). RESULTS: Mass vaccination significantly reduced the number of persistently infected pigs at 127 DPI. Vaccination did not eliminate wild-type PRRSV; administration of 2 or 3 doses of modified-live virus vaccine reduced viral shedding after 97 DPI. Previous exposure to wild-type and vaccine virus reduced clinical signs and enhanced growth following heterologous challenge but did not prevent infection. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest that therapeutic vaccination may help to reduce economic losses of PRRSV caused by infection; further studies to define the role of modified-live virus vaccines in control-eradication programs are needed.

Impact of a modified-live porcine reproductive and respiratory syndrome virus vaccine intervention on a population of pigs infected with a heterologous isolate.

The objectives of this study were to evaluate the effects of a therapeutic vaccine intervention with a modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on the dynamics of a heterologous viral infection in a population of pigs, and to determine the clinical and virological response of previously exposed and vaccinated pigs against a second virulent heterologous challenge. A population of 320 pigs were infected with a field isolate, PRRSV MN-30100, alone or followed by Ingelvac PRRS MLV vaccine administered one to three times at 30 days intervals beginning 1 week after infection. Vaccine intervention reduced the duration of viral shedding, but did not reduce the viral load in tissues or the proportion of persistently infected pigs. A different and highly virulent field isolate, MN-184, was then given as a heterologous viral challenge at 97 days after first exposure. Previously infected and vaccinated pigs showed a significant reduction in clinical signs and enhanced weight gain after the highly virulent challenge with PRRSV MN-184, but infection with and shedding of the challenge isolate were not prevented.

Further evaluation of alternative air-filtration systems for reducing the transmission of Porcine reproductive and respiratory syndrome virus by aerosol.

The purpose of this study was to compare 4 methods for the reduction of aerosol transmission of Porcine reproductive and respiratory syndrome virus (PRRSV): high-efficiency particulate air (HEPA) filtration, 2x-low-cost filtration, bag filtration, and use of a filter tested against particles derived from dioctylphthalate (DOP). The HEPA-filtration system used a prefilter screen, a bag filter (Eurovent [EU] 8 rating), and a HEPA filter (EU13 rating). The low-cost-filtration system contained mosquito netting (prefilter), 2 fiberglass furnace filters, and 2 electrostatic furnace filters. Bag filtration involved the use of a filter rated EU8 and a minimum efficiency reporting value (MERV) of 14. The 95%-DOP, 0.3-microm-filtration system involved a pleat-in-pleat V-bank disposable filter with a 95% efficiency rating for particles 0.3 microm or greater in diameter and ratings of EU9 and MERV 15. No form of intervention was used in the control group. The experimental facilities consisted of 2 chambers connected by a 1.3-m-long duct containing the treatments. Recipient pigs, housed in chamber 2, were exposed to artificial aerosols created by a mechanically operated mister containing modified live PRRSV vaccine located in chamber 1. Aerosol transmission of PRRSV occurred in 0 of the 10 HEPA-filtration replicates, 2 of the 10 bag-filtration replicates, 4 of the 10 low-cost-filtration replicates, 0 of the 10 95%-DOP, 0.3-microm-filtration replicates, and all 10 of the control replicates. Using a similar approach, we further evaluated the HEPA- and 95%-DOP, 0.3-microm-filtration systems. Infection was not observed in any of the 76 HEPA-filtration replicates but was observed in 2 of the 76 95%-DOP, 0.3-microm replicates and 42 of the 50 control replicates. Although the difference between the 95%-DOP, 0.3-microm and control replicates was significant (P < 0.0005), so was the level of failure of the 95%-DOP, 0.3-microm system (P = 0.02). In conclusion, under the conditions of this study, some methods of air filtration were significantly better than others in reducing aerosol transmission of PRRSV, and HEPA filtration was the only system that completely prevented transmission.

The Liverbeads as a tool for the comet assay.

Liverbeads, cryopreserved hepatocytes entrapped within an alginate matrix, were examined for their relevance in the comet assay. It was estimated by their capacity to activate the indirectly acting mutagens, cyclophosphamide (CP), benzo[a]pyrene (BP), dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (2-AAF), into DNA reactive metabolites. The comet assay performed in alkaline condition is a sensitive method for detecting strand breaks at the level of individual cells and allows use of quiescent cells. Experimental conditions as treatment time, cell density, beads dissociation and viability were investigated. Significant statistical positive results assessed by the tail extent moment (TEM) were observed with both human and rat Liverbeads after 12h duration incubation compared to metabolic non-competent cells, HeLa S3. Due to the maintenance of specific functions assessed by the observed capacity to metabolize xenobiotics, Liverbeads represent a suitable tool system, easy to handle, for the detection of promutagens using the comet assay.